P409: NPM1 MUTATED SAMPLES ARE ESPECIALLY VULNERABLE WHEN TARGETING RAC1 IN PATIENT-DERIVED AML CELLS IN VITRO
Authors: A. Hemsing, K. P. Rye, K. J. Hatfield, H. Reikvam
Journal: HemaSphere (2022)

Background: Ras-related C3 botulinum toxin substrate 1 (Rac1) is a GTPase signaling molecule known to be overexpressed in many solid cancers and leukemia. Rac1 plays a part in several pathways regulating, e.g., cell migration, adhesion, and cell proliferation. Recent studies have revealed activation and widespread implication of the Rac1 molecule in acute myeloid leukemia (AML), and in vitro Rac1 inhibition in AML cell lines induces apoptosis and improves chemosensitivity. Aims: We studied the in vitro effect on proliferation, apoptosis, and cytokine release of five RAC1 inhibitors in a cohort of heterogeneous, consecutive patient-derived AML cells at diagnosis. We aimed to map the antileukemic effect in a relatively large AML cohort to identify potential subgroups of AML patients that could be particularly vulnerable to RAC1 inhibition. Methods: 79 patient-derived AML cell samples from diagnosis were thawed after preservation in liquid nitrogen and cultured at standard in vitro methods with the Rac1-inhibitors ZINC69391, ITX3, EHOP-16, 1A-116, and NSC23766. Cell proliferation was measured by the 3H-thymidine incorporation assay, and apoptosis was measured by the Annexin V/Propidium Iodine apoptosis assay for flow cytometry. Assessment of cytokine profiles in culture media was done with Luminex multiplex immunoassay. Results: Measured IC50 values for each compound were as follows; ZINC69391 23 µM (95% CI 15 -29 µM), ITX3 24 µM (15 – 40µM), EHOP-16 3 µM (1,8 – 3,9 µM), 1A-116 10 µM. (8-11 µM) and NSC23766 12 µM (9-15 µM). All five Rac1 inhibitors resulted in an overall antiproliferative effect, tested at dosage around IC80. Using bioinformatical classification, we observed that high versus intermediate/low antiproliferative effect was more common in NPM1 mutated (Fischer’s exact test, p=0.002) and CD34 negative (p=0.008) patient samples. The differences were insignificant regarding the presence of FLT3 mutation or according to cytogenetics, FAB classification, gender, and age. All five compounds did induce significant apoptosis and necrosis compared to untreated control by Wilcoxon signed-rank test (p< 0.0001) at dosages around IC40 and IC80. For NSC23766 only, the presence of NPM1 mutation was associated with more severe apoptosis and necrosis (p=0.01). The presence of FLT3 mutation or CD34 negativity or positivity did not influence the depth of apoptosis and necrosis. Presence of NPM1 mutation was associated with reduced viability after treatment with EHOP-16 (p=0.025), ITX3 (p=0.047) and NSC23766 (p=0.005). CD34 negativity was significantly associated with reduced viability after treatment with NSC23766 (p=0.006). Finally, Rac1 inhibition significantly reduced the cytokine release of several cytokines crucial for leukemogenesis, with the strongest effects observed for Rac1 inhibitors 1A-116 and NSC23766 (Figure 1, Euclidean clustering). Image:Summary/Conclusion: Our results suggest potent and significant antiproliferative and antiapoptotic effects of Rac1 inhibition in primary AML samples. Interestingly, patients harboring the NPM1 mutation seem significantly more vulnerable to such inhibition. Our findings support further research regarding the role of Rac1 and its pharmacological inhibition in AML.